It is well documented that therapeutic performance of exogenously administered proteins is compounded by problems of poor pharmacokinetics and pharmacodynamics, and immunogenicity when administered to humans. A short half-life in the systemic circulation resulting from rapid clearance from the blood, degradation by plasma proteases, or inactivation by specific inhibitors impairs efficacy. Repeated parenteral administration of a heterologous (non-host) protein generally results in an immunogenic response that accelerates the clearance of the protein from the blood. Such administration may also lead to the development of hypersensitivity. If a human-derived protein is produced by a cell other than the human cell in which it is normally expressed, it may lack the proper post-translational modifications, and may in its unmodified form be immunogenic. In these instances, severe allergic reactions are common and anaphylactic death may ensue.
There has been a number of attempts to covalently attach small molecules to peptides, proteins and other biosurfaces for the purpose of improving therapeutic performance. The theory is that conjugation with poorly immunogenic substrates would mask determinant sites on the protein surface responsible for immunogenicity. Moreover, the theory is that conjugates having increased size would be expected to exhibit lower clearance rates and corresponding extended circulating life following infusion. Likewise, conjugation of small molecules and polymers to antigenic surfaces would mask protein-absorption sites on the surface of a material, device, or instrument.
Polysaccharides such as dextran exemplify polymers that may be covalently attached to peptides, proteins or surfaces for the purpose of improving their therapeutic performance. A polysaccharide that is a reducing sugar contains an aldehyde group present as a hemiacetal. In its hemiacetal form the polysaccharide is too unreactive to be practical for surface modification. Also, the size of the polysaccharide interferes sterically with reaction at this site. In addition, if the polysaccharide is negatively charged, as is for example, dextran sulfate, the negative charges can effect electrostatic hindrance to their utility as surface modification reagents. For these reasons, polysaccharides are generally regarded as not useful for surface modification without additional modification to minimize or eliminate these shortcomings.
In general, conjugate-modified peptides and proteins exhibit properties which are different from those of the native protein. Conjugation has been used with varying degrees of success to alter certain physical characteristics of peptides and proteins, including size, stability, resistance to thermal or proteolytic inactivation, pH optimum, and solubility. Attachment of hydrophilic polymers to peptides or proteins has been used to enhance blood circulation half-life, alter the immunological response, protect against protease inactivation (or inappropriate protease activation), and enhance functional stability and increase solubility. However, proteins, whether native or molecularly modified, administered parenterally may also be antigenic, initiating undesirable immune responses. Repetitive administration of polymer-conjugated peptides or proteins may induce an immune response specific for the conjugated entity. Moreover, some modifications may inactivate some or all of the biological activity of the modified peptide or protein.
As an example of a protein with therapeutic utility, adenosine deaminase (ADA) catalyzes the irreversible deamination of adenosine to inosine. Inherent defects of ADA lead to abnormalities in purine nucleoside metabolism that are selectively toxic to lymphocytes and result in immune deficiency diseases. The native enzyme has limited therapeutic efficacy following its administration, because it is rapidly cleared from the systemic circulation. Adenosine deaminase has been subjected to a series of modifications in attempts to alter its clearance rate from the circulation. For example, modification of this enzyme with monomethyl polyethylene glycol (PEG) increased the circulating half-life in humans from minutes (native protein) to five days (MS Hershfield et al. New England J Med, 316: 589, 1987). However, immunological studies following repeated infusions demonstrated antibodies specific for the modified enzyme. (FF Davis et al. Advances in Parenteral Sci Tech, 4: 831-64, 1990)
Conjugation of small molecules or polymers to surfaces has altered the surface properties and performance characteristics thereof. The conjugating entity may enhance biocompatibility of the surface by weakening the interaction between plasma proteins and the surface, inhibiting deposition of undesired cells or macromolecules by altering the surface charge and the like. The net result of the enhanced biocompatibility is maintenance of functional performance and increased longevity of utility.